METHODS AND COMPOSITIONS FOR INHIBITING THROMBOGENESIS

Abstract: Thrombus or clot formation and accretion are inhibited by administering thrombin-displacing substances to a patient. The thrombin-displacing substances comprise either thrombin analogs which bind to the thrombin-binding site on fibrin or fibrin analogs which bind to a fibrin-binding site on thrombin. By displacing the thrombin from clot or thrombus, the thrombin is released into circulation where it is inactivated by endogenous anti-proteinases. The fibrin analogs which bind to the fibrin-binding site on thrombin may be linked to a second binding moiety which binds to and/or inactivates the catalytic site and/or exosite I on thrombin to further inhibit thrombosis. The ability of test substances to displace thrombin is measured by exposure of the substance to immobilized thrombin-fibrin complex.

Description: METHODS AND COMPOSITIONS FOR INHIBITING THROMBOGENESIS BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to methods and compositions for the treatment of cardiovascular disease. More particularly, the present invention relates to novel methods and compositions to inhibit thrombus formation and growth by displacing thrombin bound to fibrin in thrombus or clot. Thrombosis is a pathological manifestation of the clotting cascade in blood vessels. The clotting cascade is a complex biological process which results in the formation of clot or thrombus composed of platelets and fibrin. Thrombosis, together with atherosclerosis, can cause partial or total occlusion of the affected blood vessel, thereby leading to a number of important cardiovascular complications, including unstable angina, acute myocardial infarction (heart attack) , cerebral vascular accidents (stroke) , pulmonary embolism, deep vein thrombosis, and arterial thrombosis. These diseases are a major cause of disability and mortality in Western societies. Of particular interest to the present invention, activation of the clotting cascade results in the generation of thrombin, which is a multi-functional serine protease that mediates thrombosis by activating platelets and cleaving fibrinogen to produce fibrin. Because thrombin also induces smooth muscle cell and fibroblast proliferation and is chemotactic for leukocytes and monocytes, it may be involved in proliferative responses, such as injury-induced atherosclerosis and restenosis after angioplasty or atherectomy. Clot or thrombus which forms as a result of activation of the clotting cascade contains fibrin, platelets, and numerous other blood components. During the process of thrombin-induced conversion of fibrinogen to fibrin, thrombin is bound to fibrin where it remains active and is protected from fluid-phase thrombin inhibitors. The resulting reservoir of clot-bound thrombin causes growth of the clot by continued cleavage of fibrinogen and activation of platelets and other blood factors, such as factor V and factor VIII. Because thrombin is critical to thrombus formation, the use of agents that inhibit thrombin formation or thrombin activity for the treatment of thrombosis and thrombotic complications has long been proposed. A number of at least partially effective inhibitors have been in use for years. For example, heparin is widely used as an anticoagulant. Heparin can inhibit both thrombin formation and thrombin activity by catalyzing the activity of antithrombin III, which is a naturally occurring inhibitor of thrombin, activated factor X (factor Xa) and other activated clotting factors. Although effective at inhibiting thrombus formation in some disorders, the use of heparin has a number of limitations. The doses required are quite unpredictable, and heparin is unable to inactivate clot- bound thrombin, allowing the continued growth of thrombus mediated by thrombin within pre-existing thrombus. The inability of heparin to inactivate thrombin bound to fibrin may explain the limited efficacy of heparin in certain circumstances, particularly when heparin is used for the treatment of coronary artery thrombosis associated with heart attacks and angina. Inactivation of clot-bound thrombin may be achieved with another set of compounds including hirudin and its derivatives. Hirudin is an antithrombin substance initially extracted from the salivary glands of the leech and now produced by recombinant DNA technology. Related compounds include hirulog, which is a small synthetic analog of hirudin. Unlike heparin, hirudin and hirulog interact directly with thrombin and do not require antithrombin III to provide anticoagulant activity. While hirudin and hirulog are able to inhibit clot-bound thrombin, the inactivation appears to be reversible so that the inhibitory effect is lost when the drugs are cleared from the blood. In contrast to hirudin and hirulog, which interact with more than just the active site of thrombin, another group of compounds having even lower molecular weights, typically about 500 Daltons, act as direct thrombin inhibitors. These compounds bind exclusively to the active (catalytic) site of thrombin in a reversible or irreversible manner. One such irreversible active-site inhibitor is PPACK (D-phenylalanyl-L- prolyl-L-arginyl chloromethyl ketone) . Such inhibitors, however, generally lack sufficient specificity for thrombin and thus have questionable safety. Moreover, there is some evidence that total inhibition of thrombin with irreversible active site inhibitors could lead to excessive bleeding. For these reasons, it is desirable to identify novel methods and compositions for inhibiting thrombogenesis associated with cardiovascular disease. In particular, it would be desirable to provide methods and compositions which are capable of permanently reducing or eliminating fibrin- bound thrombin activity from existing thrombi within the vascular system. Such elimination or reduction of clot-bound thrombin should be essentially irreversible so that clot accretion will not resume after administration of a treating agent is stopped. It would be particularly desirable to develop methods and compositions which are able to inactivate thrombin within the thrombus with an agent that does not directly inhibit clotting, since such agents would not adversely affect bleeding control (hemostasis) . Such an agent could be a compound which would displace and release thrombin into blood circulation where it would be inactivated by natural antithrombin components, such as antithrombin III and heparin cofactor II. Alternatively, the methods and compositions would act to both displace thrombin from clot and inactivate it. Finally, the methods and compositions may act by binding and inactivating thrombin that remains bound to the clot. 2. Description of the Background Art Hirudin derivatives for blocking the active site of thrombin are described in U.S. Patent Nos. 5,240,913 and 5,196,404. A bifunctional antithrombotic composition which includes both a glycoprotein Ilb/IIIa inhibitory domain and a thrombin inhibitory domain is described in WO 92/10575. Peptide analogs of glycoprotein Ilia for thrombogenesis inhibition are described in WO 90/00178. Inhibitors of factor X and/or Xa are described in U.S. Patent Nos. 5,239,058 and 5,189,019 and PCT publications WO 93/09803; WO 92/04378; and WO 92/01464. Inhibitors of factors VII and/or VIII are described in U.S. Patent Nos. 5,223,427 and 5,023,236 and WO 92/06711. Platelet antiadhesives and related antibodies are described in WO 92/08472. Stubbs and Bode (1993) Thrombosis Research (59.:1-58, is a review article describing the structure and function of thrombin. Hirsh (1993) Circ. 88:I-C summarizes a lecture relating to the limitations of heparin and potential advantages of new, anticoagulants as antithrombotics. SUMMARY OF THE INVENTION The present invention comprises methods and compositions for displacing thrombin from thrombus by exposing the thrombus to a substance, other than thrombin, which binds to a thrombin-binding site on fibrin or to a fibrin-binding site on thrombin. Binding occurs with sufficient affinity to displace thrombin from the thrombus and to inhibit rebinding of thrombin which may be present in a surrounding aqueous environment, typically blood. The methods will be particularly useful for inhibiting thrombus accretion by removing active thrombin from thrombus, thereby reducing or eliminating the primary initiator of clot growth at the clot- blood interface. The released thrombin will be inactivated either by endogenous thrombin inhibitors present in circulation or by agents that both remove or displace thrombin from the clot and which further inactivate the thrombin. The present invention further comprises pharmaceutical compositions comprising or consisting essentially of the substance which binds to a thrombin-binding site on fibrin or a fibrin-binding site on thrombin with the requisite affinity. Substances which bind to fibrin may be selected from the group consisting of thrombin fragments, thrombin analogs, thrombin mimetics, antibody and antibody fragments which bind to the thrombin-binding site on fibrin, and the like. Such fibrin-binding substances will bind to the thrombin-binding site on fibrin with an affinity of at least 5xl0 4 M _1 , preferably being at least about IO 5 M "1 , more preferably being at least 5xl0 5 , or higher. Substances which bind to the fibrin-binding site on thrombin may be selected from the group consisting of fibrin fragments, fibrin analogs, soluble prothrombin factor 2 (F 2 ) from prothrombin and fragments thereof, and antibody and antibody fragments which bind to the fibrin-binding site on thrombin, fibrin mimetics, and the like. Such substances will bind to the fibrin-binding site on thrombin with an affinity of at least 10 7 M "1 , preferably being at least about 10 s M "1 , or higher. The present invention still further provides thrombin inhibitors which comprise a hybrid molecule having a first moiety which binds to the fibrin-binding site on thrombin and a second moiety which binds to and/or sterically interferes with a proteolytic (catalytic) site on thrombin and/or fibrinogen-binding exosite I, wherein the first and second moieties are spaced-apart by a distance sufficient to permit simultaneous binding of both moieties to their respective binding sites. The first moiety is selected from the group consisting of fibrin and fragments, fibrin analogs, F 2 from prothrombin and fragments thereof, antibody and antibody fragments which bind to the fibrin-binding site on thrombin, and fibrin mimetics, and will have a binding affinity of at least about IO 7 M _1 , preferably being at least about IO 8 M "1 . A second moiety intended to interact with the proteolytic (catalytic) site on thrombin may be any compound, component, or structure which binds to the proteolytic (catalytic) site on thrombin, typically being derived from known thrombin inhibitors, such as PPACK or its derivatives, hirudin, anti-thrombin antibodies, and the like. Alternatively, the second moiety could bind to a site sufficiently near the thrombin catalytic site so that said moiety, when bound, will sterically interfere with the normal catalytic activity, usually by inhibiting binding of fibrinogen. A second moiety intended for binding exosite I may be any compound, component, or structure which binds to the fibrinogen-binding site on thrombin, typically being derived from fibrinogen or known fibrinogen analogs, analogs of other thrombin substrates, such as thrombin receptor present on platelets, thro bomodulin, factor V or factor VIII, anti- thrombin antibodies, and the like. The second moiety on the hybrid thrombin inhibitor molecules may be further modified or selected to provide both the proteolytic-site binding activity and the exosite I binding activity, as described above. The hybrid molecules may be prepared by conventional chemical synthesis techniques, or in the case of polypeptides, may be prepared reco binantly as fusion proteins. The binding affinity between the second moiety and the target site (i.e. proteolytic site and/or exosite I) will be at least about IO 5 M "1 , preferably being at least about 5xl0 5 M "1 . The distance between first and second binding moieties will typically be in the range from about 5 A to 60 A, preferably in the range from about '20 A to 55 A. A further understanding of the nature and advantages of the invention will become apparent by reference to the remaining portions of the specification and drawings. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic illustration of the thrombin molecule illustrating the relative positions of the fibrin- binding site (anion-binding exosite II) , the heparin-binding site, the catalytic site, and the fibrinogen-recognition site (anion-binding exosite I) . Fig. 2A illustrates the thrombin molecule bound to fibrin, as would be the case in formed thrombus or clot. Fig. 2B illustrates the mechanism underlying the present invention where a fibrin analog or other mimetic compound (such as soluble F 2 from prothrombin or fragments thereof) has displaced fibrin at the fibrin-binding site on thrombin to release thrombin from the clot. Fig. 2C illustrates an alternative mechanism underlying the present invention where a thrombin analog or other mimetic compound has been introduced to bind to the thrombin-binding site on fibrin and to thereby displace previously bound thrombin from the clot or thrombus. Figs. 3, 4, and 5 illustrate still further embodiments of the present invention where a bivalent or trivalent molecule includes a first moiety (Ml) capable of binding to the fibrin-binding site on thrombin linked by a spacer arm (L) to a second moiety capable of binding to (a) the catalytic site on thrombin (Fig. 3) , (b) both the catalytic site and fibrinogen-binding site on thrombin (Fig. 4) , or (c) the fibrinogen-binding site on thrombin (Fig. 5). DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS The present invention provides methods and compositions for interfering with and disrupting binding between thrombin and fibrin within thrombus or clot, whereby the ability of clot-bound thrombin to catalytically promote further clot accretion is diminished or eliminated. The compositions and methods of the present invention are particularly useful for treating patients suffering from or at risk of suffering from thrombus-related cardiovascular conditions, such as unstable angina, acute myocardial infarction (heart attack) , cerebrovascular accidents (stroke) , pulmonary embolism, arterial thrombosis, deep vein thrombosis, and the like. The present invention is not limited to such uses, however, and the methods and compositions described herein may find use in other in vitro and in vivo situations whenever it is desirable to inhibit clot or thrombus accretion or promote clot or thrombus solubilization. For example, the compositions of the present invention may be used as an anticoagulant for inhibiting thrombin-induced clotting in various in vitro studies, assays, and the like. The present invention is based at least in part on the recognition that the fibrin-binding site on thrombin is distinct from the fibrinogen-binding site. That is, when thrombin is bound to fibrin, as is the case in clot or thrombus matrices, the fibrin-binding site will be occupied while the fibrinogen-binding site remains to at least some extent sterically available to bind fibrinogen and orient the bound fibrinogen for interaction with the thrombin catalytic site which promotes the conversion of fibrinogen to fibrin. Since the fibrinogen-binding site on thrombin also mediates the interaction of thrombin with substrates in addition to fibrinogen, the bound thrombin can also activate platelets and factors V and VIII, thereby locally amplifying coagulation. The bound thrombin can also activate factor XIII, which by cross-linking the fibrin and cross-linking the α 2 -antiplasmin, renders the clot more resilient to breakdown. The present invention is further based at least in part on the recognition that by binding thrombin, the fibrin clot can act as a reservoir of active thrombin that can be steadily released back into circulation where it can interact with thrombin substrates to cause further clotting before being inactivated by thrombin inhibitors. In one aspect of the present invention, thrombin is removed from fibrin to deplete the reservoir of clot-bound thrombin and inhibit subsequent clot growth. Referring to Fig. 1, the relevant binding domains of thrombin will be described. Thrombin is a trypsin-like serine protease which displays a number of activities in the clotting cascade (blood coagulation pathway) , including the catalytic conversion of soluble fibrinogen into insoluble fibrin, wherein fibrinopeptides A and B are cleaved from the amino- terminal regions of the Aα- and B/3-chains of fibrinogen, respectively. In particular, the amino-terminal ends of the Aα- or B8-chains of fibrinogen bind to exosite I on thrombin, thereby orienting the fibrinogen chain so that the scissile Arg-Gly bond is positioned in the active center of thrombin. After cleavage of the fibrinopeptide from fibrinogen, thrombin can cleave other fibrinogen chains. During this process of thrombin-mediated conversion of fibrinogen to fibrin, thrombin becomes bound to fibrin at least in part through the fibrin- binding site (anion-binding exosite II) on thrombin. As the fibrin-binding site is remote from both the fibrinogen- recognition site (anion-binding exosite I) and the catalytic site, the clot-bound or thrombus-bound thrombin remains catalytically active. In addition, the thrombin is protected from inactivation by serum anti-proteinases, possibly because conformational changes which occur upon fibrin-binding hinder the interaction of clot-bound thrombin with naturally- occurring inhibitors. Thus, the fibrin-bound thrombin in the clot or thrombus remains available to locally amplify coagulation through activation of platelets, factor V, and factor VIII, as well as through the conversion of fibrinogen to fibrin, in order to cause clot growth. In addition, the clot-bound thrombin can activate factor XIII which then both cross-links the newly-formed fibrin and covalently links α 2 - antiplasmin onto the clot, thereby rendering the thrombin less susceptible to plasmin-mediated lysis. Referring to Fig. 1 in more detail, thrombin is a globular-ellipsoid molecule having an active catalytic site on a surface thereof. The fibrinogen-recognition site (anion- exosite I) is a highly positively charged region which interacts with fibrinogen as well as with other thrombin substrates, such as the platelet thrombin receptor, factor V, and factor VIII. Exosite I also mediates the interaction of thrombin with thrombomodulin and the carboxy-terminals of hirudin and hirulog. Binding to the anion-binding exosite I is an important step in orienting substrates relative to the catalytic site in order to achieve efficient proteolytic hydrolysis of the substrates. The inventors herein have recognized that a second positively charged binding site on thrombin, designated anion- binding exosite II, is involved in the binding of thrombin to fibrin in clot and thrombus. The anion-binding exosite II is located near to or overlapping with the putative heparin- binding site on thrombin and is centered around the side chains of the Arg 93, Arg 126, Lys 236, and Lys 240 residues on the thrombin molecule and extends to the left to encompass the side chains of the Arg 97, Arg 101, Arg 165, Lys 161, Arg 173, Arg 175, Arg 233, and Lys 235 residues. This structure results in a highly electropositive surface region on the thrombin molecule which is located hear the carboxy- terminal helix of the B-chain of thrombin. In addition to heparin, the F 2 fragment of prothrombin (which contains the second prothrombin kringle) also binds to this region, and the anion-binding exosite II is masked in prothrombin, meizothro bin, prethrombin I, and meizothrombin des F 1 . Thus, the positively charged Arg and Lys residues that comprise exosite II on thrombin form salt bridges with acidic side chains of the second prothrombin kringle. The anionic motif that mediates the interaction of F 2 with exosite II is Asp- Gly-Asp-Glu-Glu (SEQ ID NO. l) . In contrast, in Lysine- binding kringles (such as those found in plasminogen or tissue plasminogen activator) , the anionic motif is Asp-X-Asp. In one aspect, the present invention relies on disrupting or interfering with binding between the fibrin- binding site on the thrombin and/or the thrombin-binding site on fibrin in order to release thrombin from thrombus or clot into a surrounding aqueous environment, usually into blood in the vascular environment. While fibrin-bound thrombin in thrombus or clot is protected from inactivation by heparin and endogenous anti-proteinases, thrombin released from clot or thrombus will become susceptible to inactivation from endogenous anti-proteinases and/or appropriate drug therapies. The methods and compositions of the present invention can provide for complete or near-complete release and displacement of clot-bound or thrombus-bound thrombin whereby thrombin- mediated amplification of coagulation is substantially inhibited or prevented. Usually, at least about 95% inhibition of thrombus-bound thrombin may be achieved, preferably being at least about 99% inhibition, and more preferably being at least about 100% inhibition. By "inhibition," it is meant that the thrombin activity is irreversibly inactivated so that the thrombin molecule cannot promote thrombus or clot formation or accretion. Referring now to Figs. 2A and 2B, a first mechanism for displacing thrombin from fibrin in clot or thrombus will be described. In clot and thrombus, thrombin T is bound to fibrin F at least in part in a manner that leaves the fibrinogen binding site FBS and the catalytic (proteolytic) site CS sterically available (Fig. 2A) to promote blood coagulation which results in thrombus and clot accretion, as described previously. In order to displace thrombin from the thrombus, a fibrin analog FA is introduced to the thrombus (typically via introduction into patient circulation) to bind to the fibrin binding site FS on thrombin T, as illustrated in Fig. 2B. The fibrin analog may be any substance that will bind to the fibrin binding site FS on thrombin in place of clot-fibrin in order to release the thrombin from thrombus into circulation. Suitable fibrin analogs include fibrin fragments, modified fibrin polypeptides, soluble F 2 from prothrombin and fragments thereof, fibrin mimetics (small molecules designed to bind to the fibrin-binding site on thrombin in preference to native fibrin) , antibodies and antibody fragments specific for the fibrin-binding site on thrombin, or any other substance capable of entering the thrombus/clot matrix and disrupting binding between clot-bound fibrin and thrombin in order to release thrombin from the thrombus. The fibrin analog will have a binding affinity for fibrin-binding site on thrombin which is usually greater than the native binding affinity between fibrin and thrombin in thrombus. The native binding affinity is about IO 6 M" 1 , and the binding affinity of the fibrin analog will usually be at least IO 7 M" 1 , preferably being at least IO 8 M -1 , or higher. Generally, fibrin analogs, will be utilized at lower concentrations than thrombin analogs since there are fewer thrombin molecules to block. To maintain the desired blocking and inhibition, it is desirable to utilize fibrin analogs having a higher binding affinity than the native affinity between thrombin and fibrin in clot. In addition to such threshold binding affinity, the fibrin analogs should display binding kinetics which permit displacement of thrombin from the thrombus in a rapid manner. Additionally, suitable fibrin analogs will have a relatively long half-life in the circulation, preferably being at least as great as the half-life of thrombin released from the thrombus, preferably being longer than that of thrombin. It will be appreciated, however, that the only essential requirement for fibrin analogs according to the present invention is the ability to displace thrombus-bound thrombin and release said thrombin into circulation where it will be inactivated by endogenous anti-proteinases. In particular, fibrin analogs may be peptidic, e . g. , comprising or consisting essentially of the thrombin-binding site on fibrin. The thrombin-binding site on fibrin appears to be located on the central E-domain of fibrin, as described in Mosesson (1990) J. Lab. Clin. Med. 116:8-17; Vali et al. (1988) Biochemistry 27:1956-1963; and Kaminski and McDonaugh (1987) Biochem. J. 242:881-887. Such fibrin fragments and peptidic analogs can be prepared by conventional solid phase synthesis or recombinant techniques, as are well described in the art. Suitable solid phase synthesis techniques are based on the sequential addition of amino acids to a growing chain on a solid-phase substrate, as first described in Merrifield (1963) J. Am . Chem . Soc . 8J5:2149-2156. Commercial systems for automated solid-phase synthesis are now widely available from suppliers, such as Applied Biosystems, Inc., Foster City, California. Recombinant polypeptide production techniques are widely described in the technical and scientific literature. See, for example, Molecular Cloning: A Laboratory Manual , Sambrook et al., Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), Vols. 1-3. Alternatively, suitable fibrin analogs may be prepared from digests of fibrin or fibrinogen. The substance which serves as the fibrin analog may also be an antibody or antibody fragment which binds to the fibrin-binding site on thrombin, or a small molecule mimetic (typically having a molecule weight below 3 kD, usually below 2 kD, and often below 1 kD) which displays similar or enhanced binding characteristics relative to intact fibrin. Such antibodies may be prepared by employing thrombin, or a thrombin fragment comprising the fibrin-binding site, as an immunogen in conventional techniques for preparing polyclonal or monoclonal antibodies. Such techniques are well described in the scientific and patent literature. See, for example. Antibodies: A Laboratory Manual , Harlow and Lane, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1988) . The preparation of small molecule mimetics is further described below. Other substances which bind to the fibrin-binding site on thrombin include prothrombin F 2 and fragments thereof. These substances can be prepared by solid-phase synthesis or recombinant techniques, as described above. Small molecule mimetics of these substances would also be suitable. A second mechanism for thrombin displacement according to the present invention is illustrated in Fig. 2C, where a thrombin analog TA is introduced to thrombus or clot to disrupt binding between thrombin T and fibrin F. The thrombin analog TA will possess a fibrin-binding site FS having an affinity which is at least equal to, and preferably greater than, the native binding affinity between thrombin and fibrin as described above. Thus, the thrombin analog will preferably bind to fibrin with affinity of at least 5xl0 4 M "1 , preferably being at least 10 5 M -1 , and more preferably being at least 5xl0 5 M -1 . The thrombin analogs may possess a lower binding affinity than the fibrin analogs since the thrombin analogs will be used at higher concentrations (to block the excess thrombin-binding sites that may be present on fibrin) . Principles of mass action dictate that equivalent displacement will be achieved under both circumstances. The thrombin analog will also possess adequate binding kinetics and serum survivability, as described above for the fibrin analog. Suitable thrombin analogs include thrombin fragments active-site-blocked thrombin, and prothrombin 2 and its derivatives, each of which contains a fibrin-binding site, as described above. The thrombin analogs may comprise antibodies and antibody fragments which are capable of binding to the thrombin-binding site on fibrin. The preparation of polypeptide analogs and antibodies and antibody fragments may be carried out by the conventional techniques described above in connection with preparation of the fibrin analogs. Small molecule mimetics of both the fibrin analogs and thrombin analogs can be achieved through the use of techniques known to those working in the area of drug design. Such methods include, but are not limited to, self-consistent field (SEF) analysis, configuration interaction (CI) analysis, and normal mode dynamics computer programs, all of which are now readily available. See, Rein et al., Computer-Assisted Modeling of Receptor-Ligand Interactions , Alan Liss, New York (1989) and Navia and Marcko, "Use of Structural Information in Drug Design," in Current Opinion in Structural Biology, Vol. 2, No. 2, pages 202-210, 1992. The preparation of compounds identified by these techniques will depend on their structure and other characteristics and may normally be achieved by standard chemical synthesis methods, as described in available texts, such as Furniss et al., Vogel ' s Textbook of Practical Organic Chemistry. , John Wiley & Sons, New York 1992 and Larock, Comprehensive Organic Transformation , VCH Publishers, Inc., New York 1989. Compositions according to the present invention will also include multivalent, usually bivalent or trivalent, thrombin inhibitors comprising a hybrid molecule having a first moiety which binds to a fibrin-binding site on thrombin and a second moiety which binds to (or near) the catalytic (proteolytic) site and/or exosite I (the fibrinogen-binding site) on thrombin. The first moiety may comprise any of the thrombin-binding substances (i.e., fibrin analogs, prothrombin F2, or fragments, analogs, or mimetics thereof) described above, while the second moiety will be selected to bind to and sterically hinder and/or inactivate the catalytic site and/or to bind to and sterically hinder exosite I on thrombin. Suitable second moieties include (a) substances which are capable of binding to and/or sterically hindering the catalytic site (usually binding to the site with an affinity of at least 10 5 M "1 , preferably being at least 5xl0 5 M "1 ) , and (b) substances which are capable of binding to the proteolytic (catalytic) site and exosite I on thrombin (usually binding to the sites within avidity of at least about IO 5 M "1 , preferably being at least 5xl0 5 M" 1 ) , and (c) substances which are capable of binding to exosite I without binding to the catalytic site (usually binding to exosite I with an affinity of at least IO 6 M" 1 , preferably being at least IO 7 M "1 ) . Exemplary second moieties which bind to or near the catalytic site include PPACK ([DJPhe-Pro-Arg- X) , antibodies and antibody fragments which bind at or near the thrombin catalytic site, and other peptides selected or designed to have the requisite binding characteristics. Small molecules (typically below 2 kD and usually below 1 kD) may also be designed to bind to or sufficiently near to the catalytic site to provide the desired inhibition of catalytic activity. Exemplary second moieties which bind to both the catalytic site and exosite I include hirudin and hirudin analogs and fragments (e.g., hirulog), antibodies and antibody fragments which bind at or near the catalytic site and exosite I, and other peptides selected or synthesized to have the requisite binding characteristics. Exemplary second moieties which bind to or near exosite I include fibrinogen fragments and analogs, hirugen, and other analogs and mimetics of the carboxy-terminus of hirudin, analogs of thrombin substrate, such as the platelet thrombin receptor, factor V, factor VIII, or analogs of thro bomodulin, which is a thrombin-binding receptor found on endothelial cells. As illustrated in Figs. 3 through 5, bivalent molecules BV according to the present invention comprises a first moiety Ml and a second moiety M2 joined by a linking region L. The linking region or group is selected to provide the necessary covalent bridge between the first binding moiety and the second binding moiety. Frequently, the linking region will be derived from a heterobifunctional compound having a reactive group at one end which is capable of binding to the first binding moiety and a second reactive group which is capable of binding to the second binding moiety. Alternatively, the linking region may be synthesized together with either the first binding moiety, the second binding moiety, or both, and then will include only a single reactive functionality for covalent binding therebetween. The nature of the linking region is not critical, but it should provide a sufficient spacing and flexibility between the two binding moieties so that the first binding moiety Ml may bind to the fibrin-binding site FS on thrombin while the second binding moiety M2 binds to (or sufficiently near to sterically hinder activity of) the catalytic site and/or exosite I, as illustrated. The length of the linking region will usually between about 5 A and 60 A, preferably between about 10 A and 30 A between the fibrin-binding site and the catalytic site, or 25 A to 50 A between the fibrin- binding site and exosite I. The linking region should be resistant to degradation when administered to a patient as part of a therapy according to the present invention and should further not contribute to nonspecific adhesion of the hybrid molecule, i.e., adhesion or binding to other than the fibrin-binding site, the catalytic site, and/or exosite I on thrombin. It will be appreciated that the hybrid molecules should also display no or minimum immunogenicity so that the drugs they comprise may be administered to a patient over an extended period of time.* In addition to the linking of moieties through the use of heterobifunctional linking compounds, it will be possible to prepare polypeptide bivalent molecules via conventional recombinant techniques. In particular, the preparation of fusion proteins where the first and second moieties are represented by different genes and/or gene fragments joined together in proper reading frame with a suitable promoter is possible. For example, the cDNA encoding prothrombin F2 or fragment thereof could be linked with the cDNA encoding hirudin or suitable hirudin fragment. Expression of the resulting hybrid DNA molecule would provide a fusion protein that interacts with both the fibrin-binding site and the catalytic site on thrombin, and optionally exosite I, as well. Such methods are described in detail in Sambrook et al., supra . The fibrinogen analogs, thrombin analogs, and multivalent molecules, of the present invention will be incorporated as components in pharmaceutical compositions useful to treat the cardiovascular conditions described above. Such compositions will also be useful in conjunction with conventional thrombolytic treatments, e.g., the administration of tissue plasminogen activator (tPA) , streptokinase, and the like, as well as with intravascular intervention, such as angioplasty, atherectomy, and the like. Suitable pharmaceutical compositions will contain a therapeutic amount of the thrombin-displacing substance of the present invention present in a pharmaceutically acceptable carrier. By a "therapeutic amount" it is meant that sufficient thrombin-displacing substance will be present in order to promote thrombin dissolution and/or inhibit thrombus accretion when treating a thrombus-related cardiovascular condition, such as those described above. Typically, the thrombin-displacing substance will be present in a pharmaceutical composition at a concentration from about 0.01 μg per dose to 10 mg per dose, usually being in the range from about 1 μg per dose to l mg per dose. Daily dosages may also vary widely, depending on the activity of the particular thrombin-displacing substance, usually being between about 1 μg per kg of body weight to about 10 mg per kg of body weight per day, usually being from about 10 μg per kg of body weight to about 1 mg per kg of body weight per day. The pharmaceutically acceptable carrier can be any compatible, non-toxic substance suitable to deliver the thrombin-displacing substance to the patient. Sterile water, alcohol, fats, waxes, and inert solids, may be used as the carrier. Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical compositions. Such compositions will be suitable for oral, nasal, respiratory, or parenteral administration, preferably being suitable for parenteral administration, i.e., subcutaneous, vascular, and intravenous administration. It may also be possible to deliver the thrombin-displacing substances of the present invention via transdermal administration. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: HAMILTON CIVIC HOSPITALS RESEARCH DEVELOPMENT, INC. (B) STREET: 711 Concession Street (C) CITY: Hamilton (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): L8V 1C3 (ii) TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR INHIBITING THROMBOGENESIS (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/175,211 (B) FILING DATE: 27-DEC-1993 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Glu Asp Glu Glu 1

Claims: WHAT IS CLAIMED IS: 1. A method for displacing thrombin bound to fibrin in thrombus, said method comprising exposing the thrombus to a substance other than active thrombin in an aqueous environment, which substance binds to a thrombin- binding site on fibrin or a fibrin-binding site on thrombin with sufficient affinity to displace thrombin from the thrombus and inhibit thrombin in the aqueous environment from binding to fibrin in the thrombus. 2. A method as in claim 1, wherein the substance binds to fibrin and is selected from the group consisting of thrombin fragments, thrombin analogs, and antibody and antibody fragments which bind to the thrombin-binding site on fibrin. 3. A method as in claim 2, wherein the affinity is at least 5xl0 4 M "1 . 4. A method as in claim 1, wherein the substance binds to thrombin and is selected from the group consisting of fibrin fragments, fibrin analogs, soluble F 2 fragments from prothrombin, and antibody and antibody fragments which bind to the fibrin-binding site on thrombin. A method as in claim 4, wherein the affinity is at least IO 7 M" 1 , 6. A method for inhibiting thrombus accretion, said method comprising exposing the thrombus to a substance other than active thrombin which binds to a thrombin-binding site on fibrin or a fibrin-binding site on thrombin with sufficient affinity to displace thrombin from the thrombus. 7. A method as in claim 6, wherein the substance binds to fibrin and is selected from the group consisting of thrombin fragments, thrombin analogs, thrombin mimetics, and antibody and antibody fragments which bind to the thrombin- binding site on fibrin. 8. A method as in claim 7, wherein the affinity is at least 5xl0 4 M "1 . 9. A method as in claim 6, wherein the substance binds to thrombin and is selected from the group consisting of fibrin fragments, fibrin analogs, fibrin mimetics, soluble F 2 fragments from prothrombin, and antibody and antibody fragments which bind to the fibrin-binding site on thrombin. 10. A method as in claim 9, wherein the affinity is at least 10 7 M "1 . 11. A method for treating a thrombus-related condition in a patient, said method comprising administering to the patient a substance which binds to a thrombin-binding site on fibrin or a fibrin-binding site on thrombin having an affinity and in an amount sufficient to displace thrombin from thrombus. 12. A method as in claim 11, wherein the substance binds to fibrin and is selected from the group consisting of thrombin fragments, thrombin analogs, and antibody and antibody fragments which bind to the thrombin-binding site on fibrin. 13. A method as in claim 12, wherein the affinity is at least 5xl0 4 M "1 . 14. A method as in claim 11, wherein the substance binds to thrombin and is selected from the group consisting of fibrin fragments, fibrin analogs, soluble F 2 fragments from prothrombin, and antibody and antibody fragments which bind to the fibrin-binding site on thrombin. 15. A method as in claim 14, wherein the affinity is at least IO 7 M "1 . 16. A method as in claim 11, wherein the displaced thrombin is released into blood circulation where it is inactivated by endogenous inhibitors including antithrombin III, heparin cofactor II, and other thrombin inhibitors. 17. A pharmaceutical composition comprising a substance which binds to a thrombin-binding site on fibrin or a fibrin-binding site on thrombin with an affinity sufficient to displace thrombin from thrombus in an aqueous environment under normal physiologic conditions, present in a pharmaceutical carrier. 18. A pharmaceutical composition as in claim 17, wherein the substance binds to fibrin and is selected from the group consisting of thrombin fragments, thrombin analogs, and antibody and antibody fragments which bind to the thrombin- binding site on fibrin. 19. A pharmaceutical composition as in claim 18, wherein the affinity is at least 5xl0 4 M "1 . 20. A pharmaceutical composition as in claim 17, wherein the substance binds to thrombin and is selected from the group consisting of fibrin fragments, fibrin analogs, soluble F 2 fragments from prothrombin, and antibody and antibody fragments which bind to the fibrin-binding site on thrombin. 21. A pharmaceutical composition as in claim 20, wherein the affinity is at least IO 7 M "1 . 22. A thrombin inhibitor comprising a hybrid molecule having a first moiety which binds to a fibrin-binding site on thrombin and a second moiety which binds a proteolytic site and/or fibrinogen-binding site (exosite I) on thrombin, wherein the first and second moieties are spaced-apart by a distance sufficient to permit simultaneous binding of both moieties to their respective binding sites. 23. A thrombin inhibitor as in claim 22, wherein the first moiety is selected from the group consisting of fibrin fragments, fibrin analogs, soluble F 2 fragment from prothrombin, and antibody and antibody fragments which bind to the fibrin-binding site on thrombin. 24. A thrombin inhibitor as in claim 23, wherein the affinity is at least IO 7 M" 1 . 25. A thrombin inhibitor as in claim 22, wherein the second moiety binds to the proteolytic site and is selected from the group consisting of PPACK, PPACK analogs, PPACK fragments, and antibodies to the proteolytic site on thrombin. 26. A thrombin inhibitor as in claim 25, wherein the second moiety binds to or near the proteolytic site with an affinity of at least 10 5 M" 1 . 27. A thrombin inhibitor as in claim 22, wherein the second moiety binds to both the proteolytic site and exosite I and is selected from the group consisting of hirudin, hirudin analogs, hirudin fragments, and antibodies to the proteolytic site and exosite I. 28. A thrombin inhibitor as in claim 27, wherein the second moiety binds to the proteolytic site and exosite I with an avidity of at least IO 5 M "1 . 29. A thrombin inhibitor as in claim 22, wherein the second moiety binds to exosite I and is selected from the group consisting of fibrinogen, factor V, factor VIII, the platelet thrombin receptor, soluble thrombomodulin and fragments and analogs thereof, the carboxy-terminus of hirudin and fragments and analogs thereof, and antibodies to exosite I. 30. A thrombin inhibitor as in claim 29, wherein the second moiety binds to or near exosite I with an affinity of at least IO 6 M "1 . 31. A thrombin inhibitor as in claim 22, present in a pharmaceutically acceptable carrier. 32. A thrombin inhibitor as in claim 22, wherein the first and second binding moieties are spaced-apart by a distance in the range from 5 A to 60 A.

Inventors: HIRSH JACK, WEITZ JEFFREY I, HIRSH, JACK, WEITZ, JEFFREY, I.
Applicants: HAMILTON CIVIC HOSPITALS RES, HAMILTON CIVIC HOSPITALS RESEARCH DEVELOPMENT, INC
Application year: 1994
Main classification: A61K38
IPC codes: A61K38, A61K39, A61P7, A61P9, A61P43, C07K14, C07K16, C12N9